3beta, 7, 11alpha-trihydroxy-5-pregnene-20-one and esters thereof



United States Patent Ofitice 2,702,809 Patented Feb. 22, 19553fi,7,11a-TRIHYDROXY-S-PREGNENE-ZU-ONE AND ESTERS THEREOF Herbert C.Murray, Hickory Corners, and Durey H. Peterson, Kalamazoo Township,Kalamazoo County, Mich., assignors to The Upjohn Company, Kalamazoo,Mich., a corporation of Michigan N Drawing. Application August 26, 1953,Serial No. 376,737

8 Claims. (Cl. 260397.45)

The present invention relates to the novel3fi,7,11atrihydroxy-S-pregnene-20-one and esters thereof.

These novel compounds may be represented by the following formula:

HO OR wherein R is hydrogen or a hydrocarbon-carboxylic acyl radical.The 35,7,1la-trihydroxy-5-pregnene-20-one may be prepared by subjectingpregnenolone (3p-hydroxy-5- pregnene-ZO-one) to the oxygenating actionof a culture of fungus of the order Mucorales as set forth in ourapplication, of which the present application is a continuation-in-part,Serial No. 180,496, filed August 19, 1950, now abandoned, Serial No.272,944, filed February 23, 1952, issued July 8, 1952 as Patent No.2,602,769, and Serial No. 296,741, filed July 1, 1952, now abandoned.

It is an object of the present invention to prepare the novel35,7,11a.-trihydroxy-5-pregnene-20-one and esters thereof. Other objectswill be apparent to those skilled in the art to which this inventionpertains.

The thermostable compounds of the present invention have demonstratedpharmacological properties such as anti-hypertensive anti-estrogenic,anti-testoid, anti-folliculoid, anti-progesterone, and anaestheticactivities. Furthermore, the 35,7,11a-trihydroxy-5-pregene-20-one hasadditional value as a chemical intermediate, for example,35,7,11a-trihydrOXy-S-pregnene--one may be reduced with lithium aluminumhydride in tetrahydrofurane to give 35,7,11a,20-tetrahydroxy-5-pregnene,which may be oxidized with manganese dioxide in chloroform and at roomtemperature to 3,8,11 a,20-trihydroxy-5-pregnene-7- one. Reduction ofthis ketotriol by the Wolfi-Kishner method results in3,8,11(1,2O-trihydroxy-S-pregnene which may be oxidized with chromicacid in acetic acid solution to 1 l-ketoprogesterone.

The following examples are illustrative of a process for the productionof the products of the present invention but are not to be construed aslimiting.

Example 1 .-3,6,7,11a-trihydroxy-5-pregnene-20-0ne A medium was preparedof twenty grams of Edamine enzymatic digest of lactalbumin, three gramsof corn steep liquor and fifty grams of technical dextrose diluted toone liter with tap water and adjusted to a pH of 4.3 to 4.5. Eightliters of this sterilized medium was inoculated with Rhizopus arrhizus,ATCC 11145, and incubated for nineteen hours at a temperature of 28degrees centigrade using a rate of aeration and stirring such that theoxygen uptake was 6.3 to 7 millimoles per hour per liter of NazSOsaccording to the method of Cooper, Fernstrom and Miller, Ind. Eng.Chem., 36, 504 (1944). To this medium containing a nineteen-hour growthof Rhizopus arrlzizus was added two grams of pregnenolone(3fi-hydroxy-5-pregnene-20-one) in twenty milliliters of acetone toprovide a suspension of the steroid in the culture. After an additional75-hour period of incubation under the same conditions of temperatureand aeration, the beer and mycelium were extracted and concentrated. Themycelium was filtered, washed twice, each time with a volume of acetoneapproximately equal to the volume of the mycelium and extracted twice,each time with a volume of methylene chloride approximately equal to thevolume of the mycelium. The acetone and methylene chloride extractsincluding solvent were added to the beer filtrate. The mixed extractsand beer filtrate were extracted successively with four liters ofmethylene chloride and then with three two-liter portions of methylenechloride. The combined methylene chloride extracts were washed with twoone-tenth by volume portions of a two percent aqueous solution of sodiumbicarbonate and then with two one-tenth by volume portions of water.After drying the methylene chloride extract with about three to fivegrams of anhydrous sodiumsulfate per liter of solvent and filtering, thesolvent was removed by distillation leaving 3.3162 grams of crystallineresidue. This residue was dissolved in 200 milliliters of benzene andchromatographed over grams of alumina (acid washed, dried at degreescentigrate for four hours). The column was developed with ZOO-milliliterportions of solvents as indicated in the table.

Fractions 28 through 31 were combined, dissolved in ten milliliters ofmethanol, and concentrated until crystallization ensued. A few drops ofwater were added, the mixture was cooled, and the crystals wereseparated. Three recrystallizations from methanol, adding water tofacilitate crystallization, or methanol-carbon tetrachloride yielded 375milligrams of 35,7,1la-trihydroxy-5-pregnene- 20-one, melting point 247to 248 and 250 to 255 degrees centigrade, [111 of minus 41 degrees(1.086 in methanol). Structure was verified by infrared spectra.

Analysis.Percent calculated for C21H32O4: C, 72.38; H, 9.25. Found: C,72.34; H, 9.13.

Esters of 35,7,11a-trihydroxy-S-pregnene-ZO-one are prepared accordingto various acylation procedures, as by reaction with ketene, ketenes ofselected acids, selected acids, acid anhydrides, or acid chlorides, inan organic solvent such as pyridine or the like. The thus-describedacylation process is productive of the mono-, diand triesters of3,B,7,11a-trihydroxy-5-pregnene-20-one although in differentproportions, depending upon the proportions of acylating agent to35,7,11a-trihydroxy-5-pregnene-20- one. Using one molar equivalent ofacylating agent to said steroid produces predominantly the mono-acylatedproduct, about two molar equivalents is productive of predominantly thedi-ester whereas with about three molar equivalents or more the mainproduct is the triester of 3;3,7,11a-trihydroxy-S-pregnene-20-one.

Example 2 .3B,] 1 a-diacetoxy-7-hydroxy-5-pregnene- 20-0ne A47-rnilligram sample of 35,7,11a-trihydroxy-5-pregnene-ZO-one wasdissolved in six milliliters of acetic anhydride and four milliliters ofpyridine. The mixture was maintained at room temperature for fiftyhours, thereupon diluted to 100 milliliters with water and extractedwith ninety-, forty-, and forty-milliliter portions of ether. Thecombined ether extracts were washed with fifteen milliliters of water,twice with twenty-milliliter portions of normal hydrochloric acid,fifteen milliliters of water, three times with thirty-milliliterportions of seven percent sodium bicarbonate solution and three timeswith 25- milliliter portions of water. The ether solution was dried overanhydrous sodium sulfate and evaporated by a stream of air. The oilyresidue crystallized from 0.2 milliliter of methanol, and was twicerecrystallized from two milliliters of acetone to which ether was addeddrop by drop until crystals of 3&1la-diacetoxy-7-hydroxy-5-pregnene-ZO-one appeared and were removed by filtration.

Example 3.3/3,I1 e-dipropionyloxy-7-hydroxy-5- pregnene-ZO-one In thesame manner as given in Example 2, the 35,110;-dipropionyloxy-7-hydroxy-5-pregnene-20-one is prepared using theequivalent proportion of propionic anhydride in place of aceticanhydride.

Example 4.-3B,7,1 1 m-triacetOxy-S-pregnene-ZO-One Otherwise in the samemanner as given in Example 2, 35,7,11a-triacetoxy-5-pregnene-20-one isprepared uslng six equivalents of acetic anhydride.

Example 5 .3 6,7,1 1 a-tripropionyloxy-5-pregnene-20- one Otherwise inthe same manner as given in Example 2,35,7,11u-tripropionyloxy-5-pregnene-20-one is prepared using sixequivalents of propionic anhydride.

Other representative esters of 3,8,7,11a-trihydroxy-5- pregnene-ZO-onewhich can be prepared in the same manner as above, include one to eightcarbon atom carboxylic acid acyloxy esters of saturated or unsaturatedaliphatic, carbocyclic, cycloaliphatic, aryl, arylalkyl, alkaryl, mono,di or polycarboxylic acids which forms ester groups such as, forexample, formyloxy, acetoxy, propionyloxy, dimethylacetoxy,trimethylacetoxy, butyryloxy, valeryloxy, hexanoyloxy, heptanoyloxy,octanoyloxy, benzoxy, phenylacetoxy, toluoyloxy, cyclopentylformyloxy,B-cyclopentylpropionyloxy, acrylyloxy, cyclohexylformyloxy, the half anddi-esters of malonic, maleic, succinic, glutaric and adipic acids, andthe like. The acids may also contain non-interfering substituents, suchas mono or poly halo, chloro, bromo, hydroxy, methoxy, and the like, ifdesired.

If a mixed ester involving three difierent acyl groups is desired the3B,7,l1oc trihydroxy 5 pregnene 20 one may be partially esterified withone acylating agent, the mono-ester esterified with an acylating agentwhich introduces a difierent acyl group, and the di-ester may then becompletely esterified with a third acylating agent which introduces athird acyl group. Thus 35-acet0xy- 7 dimethylacetoxy 11cc Bcyclopentylpropionyloxy- 5-pregnene-20-one or other mixed esters of theherein mentioned acid groups may be prepared.

TABLE Fraction Solvent be wene benzene plus 5 percent ether benzene plus10 percent other benzene plus 50 percent ether ether 7 ether plus 5percent 011013 8 ether plus 10 percent OHC13 ether plus 50 percentCECIL... chloroform CHgls plus 5 percent acetone--- n (High plus 10percent acetone.

o acetgne plus percent methanol...

acetone plus percent methanol acetone plus 50 percent methanoL. methanolIt is to be understood that the invention is not to be limited to theexact details of operation or exact compounds shown and described asobvious modifications and equivalents will be apparent to one skilled inthe art and the invention is therefore to be limited only by the scopeof the appended claims.

We claim: 1. A compound represented by the structural formula:

No references cited.

1. A COMPOUND REPRESENTED BY THE STRUCTURAL FORMULA: